ProgramDirector/PrincipalInvestigator(Last,First,Middle):Peterson,Craig,Lewis Theoverallobjectiveofourresearchistodeterminehowchromosomestructureinfluencesgene transcription,DNAreplicationandrepair,withspecialemphasisonidentifyingandcharacterizingthechromatin remodelingmachinesthatcontrolchromosomedynamics.Notably,geneticexperimentshaverevealedATP- dependentchromatinremodelingenzymesasessentialregulatorsofvirtuallyeverychromosomalprocess,and theirdysregulationleadstoavarietyofdiseases,includingcancer.Ourresearcheffortscanbeorganized intothreeinter-relatedareas:(1)MechanisticstudiesofATP-dependentchromatinremodelingenzymes;?(2) Roleofchromatindynamicsingenomestabilitypathways;?and(3)Assembly/functionofchromatinhigherorder structures.Amajorfocusofourmechanisticstudiesistocontinuetodissectthestructureand biochemicalmechanismsoftheINO80CandSWR1Cenzymes.Theseremodelingenzymescatalyze novel,ATP-dependenthistoneexchangeeventsthatcontrolthedepositionanddistributionoftheH2A.Z histonevariantwithinnucleosomesthatflankpromotersofgenestranscribedbyRNApolymeraseII,aswellas nucleosomesthatflankchromatinboundaryelements,centromeres,andreplicationorigins.Mammalian homologsofSWR1CandINO80C,includingthep400/Tip60andhINO80complexes,arekeyforproperstem cellfunction,genomestability,development,andgeneexpression.Duringthepastbudgetperiod,weidentified anovelregulatoryinteractionbetweenSWR1Candtheacetylationoflysine56ofhistoneH3(H3-K56Ac)that regulatesnucleosomedynamics,noncodingRNAexpression,andassemblyoflarge-scale,chromosome interactiondomains(CIDs)thatarerelatedtomammaliantopologically-associateddomains(TADs).These mechanisticstudieswillincludequantitative,fluorescence-basedassaystodefinestepsofthehistonedimer exchangereaction,aswellasthereconstitutionofthesemulti-subunitenzymeswithrecombinantsubunits. Studiesfromusandothersoverthepast10yearshavedemonstratedthatchromatindynamicsplayalarge roleinstabilizingthereplisomeandincontrollingvariousstepsinDNAdoublestrandbreakrepair.Ourrecent datasuggeststhatchangesinchromatindynamicscanalsoleadtodysregulationoftranscriptionwhich impactsgenomestabilitypathways.Ourresearchwilladdressseveralkeyunansweredquestions focusedongenomestabilitypathways:(1)DoRemodelersregulatethehomologysearchstepof homologousrecombinationanddotheyfunctioninconcertwithhistoneacetylases?(2)HowdoesINO80C stabilizethereplisomeandisthisroleduetotheregulationofncRNAexpression?(3)HowdoesINO80C preventncRNAexpressionfromintergenicregions?(4)DoesthehyperacetylationofH3-K56Acleadto formationofR-loopsthatdisruptreplisomefunction?(5)DoeshypoacetylationofH3-K56Acandtheresulting defectinnucleosomeassemblyalsoleadtoaberranttranscriptionduringSphasethatleadstogenome instability?Weplantocontinuetoexploitacombinationofinvitroandinvivoapproachestoaddresssuch questions. MechanisticstudiesofRemodelershaveprimarilyfocusedonsinglenucleosomesubstratesorsimple nucleosomalarrays.Invivo,theseenzymeslikelytargetnucleosomeswithinthecontextofcondensed chromatinfibers.Recently,weusedacombinationofsedimentationvelocityanalysesandAFMtodissectthe stoichiometryandsolutiondynamicsofasimpleformofyeastheterochromatinthatcontainstheprimary structuralcomponent,Sir3.Weplantoextendsuchstudieswithchromatinfibersreconstitutedwithacomplete complementofSirproteins(Sir2/3/4).Theoverallgoalwillbetocharacterizethestructureofyeast heterochromatinfibersaswellasunderstandinghowthesestructurescanbemodulatedby Remodelers.Asecondtypeofhigherorderchromosomestructureoccursthroughoutthegenome.CIDs containstronglyself-associatingnucleosomesthatspan~1-5genes,separatedbydistinctboundaryregions. LittleisknownaboutwhatcontrolsCIDorTADassemblyorwhatfunctionalrolestheyplay.Onepossibilityis thatdomainsofself-associatingnucleosomesassemblespontaneouslyadjacenttonucleosomedepleted regions(NDRs),andmutantsthatdisruptCIDseitheraffecttheefficiencyofNDRformationorincrease transcriptionthroughCIDs.Oneofourgoalsistouseagenome-wide,nucleosomereconstitutionsystem todirectlytestwhetherformationofpromoter-associated,nucleosome-depletedregionsissufficient forCIDassemblyinvitro.TheseinvitrostudieswillalsobecomplementedbygeneticanalysesofCID assembly,wherewewilleithereliminatekeyfactorsbygenedeletion,ormanipulatespecificCIDboundary regionsbyDNAalterations.Inthelongterm,understandinghowCIDsareassembledwillfacilitatestudiesto disruptthisprocessandinvestigatethefunctionalconsequencesonbothtranscriptionandgenomestability. OMBNo.0925-0001/0002(Rev.08/12ApprovedThrough8/31/2015) Page ContinuationFormatPage